A brush structure barrier fitting nucleic acid drug delivery system and a preparation method and application thereof

Abstract

The application discloses a brush-like structure barrier adaptive nucleic acid drug delivery system and a preparation method and application thereof, and belongs to the technical field of polymer chemistry and biological medicine, wherein the delivery system is prepared through click reaction or cycloaddition reaction of a poly-bis-sulfur main chain, a modified nucleic acid drug and a modified polyampholyte; the poly-bis-sulfur main chain is a random copolymer, comprising a bis-sulfur structural unit A and an acrylate structural unit B, the bis-sulfur structural unit A is formed through ring-opening polymerization of thioctic acid and is modified with a click reaction functional group or a cycloaddition reaction functional group; the modified nucleic acid drug is modified with a click reaction functional group or a cycloaddition reaction functional group on at least one base; the modified polyampholyte is modified with a click reaction functional group or a cycloaddition reaction functional group; the delivery system can endow the nucleic acid drug with the ability to break through the skin and mucus barriers, and can be used for the treatment or prevention of various diseases according to the type and quantity of the carried nucleic acid drug.

Description

Technical Field

[0001] This invention belongs to the fields of polymer chemistry and biomedicine, specifically relating to a brush-like barrier-adapted nucleic acid drug delivery system, its preparation method, and its application. Background Technology

[0002] Nucleic acid drugs, based on DNA, RNA, and their derivatives, possess significant advantages over traditional drugs due to their unique mechanism of precisely regulating disease genes. They can intervene in disease processes at the molecular level through specific mechanisms such as antisense oligonucleotides, RNA interference, exon-jumping gene editing, and DNA decoys. Compared to traditional drugs that only target symptoms, they have greater potential for radical cure, and are particularly suitable for hereditary diseases, malignant tumors, chronic inflammation, and viral infections. Furthermore, nucleic acid drugs offer extremely high design flexibility, allowing for precise customization based on the pathogenic gene sequences of different diseases to meet personalized treatment needs.

[0003] However, overcoming the skin and respiratory tract barriers to achieve precise targeted delivery of nucleic acid drugs remains a major technological bottleneck in this field. Nucleic acid molecules are highly hydrophilic, have large molecular weights, and are extremely unstable. Once inside the body, they are easily and rapidly degraded by serum nucleases, and struggle to penetrate physiological barriers such as the stratum corneum of the skin, respiratory mucosal epithelial cells, and mucus layer. Furthermore, nucleic acid drugs have short circulation times in vivo, are easily recognized and cleared by the reticuloendothelial system, lack natural targeting, and their non-specific distribution may trigger immunogenic reactions. Combined with the barrier effect of the skin and the active clearance by respiratory mucociliary tract tract mucosal tract tract, conventional delivery methods are insufficient to achieve effective enrichment and intracellular delivery of nucleic acid drugs in target tissues.

[0004] To achieve precise targeted delivery of nucleic acid drugs, traditional solutions primarily focus on two main pathways: carrier encapsulation or chemical modification of nucleic acid molecules (e.g., Chinese patent documents with publication numbers CN120661679A, CN119112789A, CN105983102A, and CN110478322A). While carriers such as liposomes and polymer nanoparticles can protect nucleic acids to some extent, their biocompatibility is poor, and they exhibit extremely poor compatibility with the skin and respiratory barriers, easily remaining on the barrier surface or triggering local inflammatory reactions, thus failing to effectively penetrate the barrier to reach target cells. Chemical modification of nucleic acids can improve stability, but requires the design of specific solutions for particular nucleic acid molecules, and excessive modification may impair the binding ability of nucleic acids to targets, leading to a significant decrease in biological activity. Existing nucleic acid drug delivery systems are mostly designed for systemic delivery or tumor tissue, with few solutions specifically adapted to the characteristics of the skin and respiratory barriers, making it difficult to meet the clinical needs of local treatment scenarios such as skin diseases and respiratory diseases.

[0005] Therefore, there is an urgent need to develop a universal nucleic acid delivery system to systematically solve the above-mentioned technical problems, in order to overcome the skin and respiratory tract barriers, achieve anti-nuclease degradation, targeted enrichment, prolong in vivo circulation time and low immunogenicity and toxic side effects, and further improve the treatment or prevention of diseases. Summary of the Invention

[0006] To address the problems of poor stability, low cellular uptake capacity, and difficulty in penetrating skin and respiratory barriers, this invention provides a brush-structure barrier-adapted nucleic acid drug delivery system.

[0007] The specific technical solution adopted is as follows: A brush-like barrier-adapted nucleic acid drug delivery system is prepared by a click reaction or cycloaddition reaction of a polydisulfide backbone, a modified nucleic acid drug, and a modified zwitterion. The polydisulfide backbone is a random copolymer, including disulfide structural unit A and acrylate structural unit B. Disulfide structural unit A is formed by ring-opening polymerization of thioctic acid and modified with click reaction functional group or cycloaddition reaction functional group R3. The modified nucleic acid drug contains 1-100 bases, at least one base is modified with a click reaction functional group or a cycloaddition reaction functional group R4, which can react with R3 of the disulfide structural unit A in a click reaction or cycloaddition reaction. The modified zwitterionic structure is as follows: ; R5 is a click reaction functional group or a cycloaddition reaction functional group, which can react with R4 modified on the nucleic acid drug in a click reaction or a cycloaddition reaction; R6 is a zwitterionic functional group, where m or n is an integer from 1 to 500; and R7 is a chain transfer reagent residue.

[0008] This invention designs a nucleic acid drug delivery system by forming a brush-like structure through click or cycloaddition reactions between a polydisulfide backbone, a modified nucleic acid drug, and a modified zwitterion. This enhances the stability of the nucleic acid drug and its ability to penetrate the skin and respiratory tract barriers. Depending on the type, quantity, and zwitterion density of the nucleic acid drug carried, it can be used in the treatment of allergic diseases, tumors, pathogenic bacteria or viral infections, or in the development of vaccines.

[0009] Furthermore, in the polydisulfide backbone, the acrylate structural unit B is a benzyl acrylate unit, a 2,2,2-trifluoroethyl acrylate unit, a linear fatty alcohol acrylate unit, an isobornyl acrylate unit, or a polyethylene glycol acrylate unit.

[0010] The structural formula of the polydisulfide backbone is as follows: ; Where x is an integer from 1 to 500, y is an integer from 1 to 2000, and z is an integer from 1 to 1000; R1 and R1' are chain transfer reagent residues; R2 is an acrylate side group, selected from any of the following structural formulas. Indicates the connection position; .

[0011] R3 is selected from any of the following structural formulas. Indicates the connection position; .

[0012] Furthermore, when the chain transfer reagent is selected from the following types of compounds, the corresponding R1 and R1' are as follows: .

[0013] The nucleic acid drug is modified with a functional group R4 at the 5′ end, 3′ end, or a middle base, which can undergo a click reaction or cycloaddition reaction with R3; R4 is selected from any of the following structural formulas. Indicates the connection position; .

[0014] Preferably, the nucleic acid drug includes, but is not limited to, a nucleic acid targeting the β subunit of the FcεRI receptor, with the nucleotide sequence: 5′-TTCACGTGTTGCCTGTGGAAAACATGAATT-3′ (SEQ ID NO.1) or 5′-TTCACCACAAATATGGCTCCCCAGAATGGA-3′ (SEQ ID NO.2), wherein one of the T bases is modified with DBCO or is directly modified with DBCO at the 5′ end.

[0015] The chemical formulas for DBCO-modified T bases or direct DBCO modification at the 5' end are as follows. Indicates the connection position; .

[0016] Furthermore, in the modified zwitterionic polyion, R5 is selected from any of the following structural formulas. Indicates the connection position; .

[0017] Furthermore, R6 is selected from any of the following structural formulas. Indicates the connection position; 。

[0018] When the polydisulfide backbone, the modified nucleic acid drug, and the modified polyzwitterion react, the molar ratio is 1:0.01-1000:0.01-10000, more preferably 1:1-1000:1-10000, and even more preferably 1:1-100:1-1000.

[0019] The present invention also provides a method for preparing the brush-structure barrier-adapted nucleic acid drug delivery system, comprising the following steps: In a solvent system (preferably water), a click reaction or cycloaddition reaction is carried out between a polydisulfide backbone and one or more modified nucleic acid drugs, followed by the addition of one or more modified zwitterions to carry out a click reaction or cycloaddition reaction, thereby preparing the brush-like barrier-adapted nucleic acid drug delivery system. When a click reaction occurs, the reaction conditions are 0-60 °C for 2-48 h; When a copper-catalyzed click reaction occurs, use cuprous salt or copper salt-ligand catalysis; When a cycloaddition reaction occurs, the reaction conditions are 25-100 °C for 1-72 h.

[0020] The synthesis method of polydisulfide backbone is as follows: under the action of an initiator, thioctic acid, acrylate monomers and chain transfer reagents are polymerized in a solvent system to prepare the first intermediate; under the action of a catalyst, the first intermediate is reacted with a compound containing R3, and after the reaction is completed, the polydisulfide backbone is prepared by post-treatment.

[0021] Preferably, the initiator is azobisisobutyronitrile, the acrylate monomer is benzyl acrylate, and the polymerization conditions for preparing the first intermediate are -20 to 80 °C for 2 to 48 h; the catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine, and the reaction conditions for the first intermediate with the R3-containing compound are -20 to 40 °C for 1 to 24 h.

[0022] The modified polyzwitterion is prepared by reacting a chain transfer reagent with a zwitterionic monomer under the action of an initiator to obtain a second intermediate; after the second intermediate is dethiocarbonylated by n-propylamine, it undergoes an addition reaction with a methacrylate or maleimide compound containing an R5 group to prepare the modified polyzwitterion.

[0023] Preferably, the chain transfer reagent can be any one of 4-cyano-4-(thiobenzoyl)valerate, 4-cyano-4-(thiobenzoyl)valerate, 2-(dodecylthio(thiocarbonyl)thio)-2-methylpropionic acid, 4-((((2-carboxyethyl)thio)thiocarbonyl)thio)-4-cyanopentanoic acid, 4-cyano-4-(((propylthio)thiomethyl)thio)valerate, 2-(ethimercaptothiocarbamoylthio)-2-methylpropionic acid, 4-cyano-4-(((ethio)thiocarbonyl)thio)valerate, etc.

[0024] The preferred reaction conditions for preparing the second intermediate are 40-100 °C and 0.5-72 h.

[0025] Preferably, the conditions for removing the thiocarbonyl group from the second intermediate are 20-40°C for 1-24 hours; and the conditions for the addition reaction between the thiocarbonyl group removed and the methacrylate or maleimide compound containing the R5 group are 20-40°C for 0.5-72 hours.

[0026] The present invention also provides the application of the brush-structure barrier-adaptor nucleic acid drug delivery system in the preparation of disease treatment products and / or disease prevention products, particularly in the application of atopic dermatitis treatment products and allergic rhinitis treatment products.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The brush-structure barrier-adapted nucleic acid drug delivery system provided by the present invention can solve the problem that existing nucleic acid delivery systems are difficult to break through the skin and respiratory tract barriers. The process route for synthesizing this nucleic acid drug delivery system has not been reported in the prior art.

[0028] (2) The brush-structure barrier-adapted nucleic acid drug delivery system provided by the present invention has universality and can be used to load different types of nucleic acid drugs. It can also improve the stability of nucleic acid drugs and the cell uptake capacity, and can be used for the treatment or prevention of various diseases such as allergic diseases, cardiovascular diseases, neurological diseases, or rare diseases.

[0029] (3) The brush-structure barrier-adapted nucleic acid drug delivery system provided by the present invention can respond to the reducing environment, realize the release of nucleic acid drugs and exert their functions.

[0030] (4) The brush-structure barrier-adapted nucleic acid drug delivery system provided by the present invention can utilize the coupling sites on the disulfide backbone to achieve the binding of other drugs or functional molecules with nucleic acid drugs, thereby achieving combined treatment and medication to further improve the treatment or prevention effect. Attached Figure Description

[0031] Figure 1The NMR spectrum of the random polymer (thioctic acid-benzyl acrylate) in Example 1 is shown.

[0032] Figure 2 The image shows the NMR spectrum of the polydisulfide backbone in Example 1.

[0033] Figure 3 The NMR spectrum of the zwitterionic monomer in Example 1 is shown.

[0034] Figure 4 The NMR spectrum of polycarboxybetaine in Example 1 is shown.

[0035] Figure 5 The NMR spectrum of the dibenzocyclooctylene-modified polycarboxylated betaine in Example 1 is shown.

[0036] Figure 6 The hydration particle size and potential of the 70% and 30% nucleic acid vectors prepared in Examples 1-2 were characterized.

[0037] Figure 7 The results show the nuclease stability characterization of the 30% nucleic acid vector prepared in Example 2.

[0038] Figure 8 The figures show the uptake of 70% and 30% of the nucleic acid vector by mouse bone marrow-derived mast cells. In Figure A, the uptake of the vector by mouse bone marrow-derived mast cells was analyzed using flow cytometry, and in Figure B, the uptake of the vector by mouse bone marrow-derived mast cells was analyzed using confocal microscopy.

[0039] Figure 9 The results show the cytotoxicity of 70% and 30% nucleic acid vectors against human umbilical vein endothelial cells, mouse fibroblasts, and mouse bone marrow-derived mast cells.

[0040] Figure 10 To study the skin penetration depth of 70% nucleic acid vectors for nucleic acid drug treatment of atopic dermatitis.

[0041] Figure 11 To conduct a pharmacodynamic study on 70% nucleic acid vectors for nucleic acid drug treatment of atopic dermatitis.

[0042] Figure 12 To study the mucus penetration of a 30% nucleic acid vector for nucleic acid drug treatment of allergic rhinitis.

[0043] Figure 13 To study the nasal mucosal penetration of a 30% nucleic acid vector for nucleic acid drug treatment of allergic rhinitis.

[0044] Figure 14To study the efficacy of a 30% nucleic acid vector for the treatment of allergic rhinitis with nucleic acid drugs, the following data were collected: A) Record of the number of mice wiping their noses; B) Record of the number of mice sneezing; C) Proportion of mice with runny noses among all mice; D) Analysis of histamine concentration in mice; E) Immunohistochemical results of nasal cavity sections from mice; and F) Staining of mucosal goblet cells using periodic acid-Schiff (PAS). Detailed Implementation

[0045] To make the objectives, features, and advantages of this invention more apparent and understandable, a detailed description is provided below through specific embodiments. Many specific details are set forth in the following description to provide a thorough understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below. Technical features in various embodiments of the invention can be combined appropriately without mutual conflict.

[0046] Unless otherwise specified, the operating methods in the following examples are generally performed under conventional conditions or as recommended by the manufacturer. Contents not described in detail in this specification are prior art known to those skilled in the art. Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.

[0047] Example 1 (1) Synthesis of random polymer (thioctic acid-benzyl acrylate)

[0048] Random copolymer poly(lipoic acid-benzyl acrylate) was synthesized by reversible addition-fragmentation chain transfer polymerization under a strictly anhydrous and oxygen-free atmosphere (argon / nitrogen protection): Lipoic acid (206.33 mg, 1.0 mmol) was weighed and dissolved in 1 mL of anhydrous tetrahydrofuran and stirred until completely dissolved; then benzyl acrylate monomer (450.56 μL, 3.0 mmol) was added and stirred until the system was homogeneous; 2-(dodecyltrithiocarbonyl)-2-methylpropionic acid (0.013 mmol) pre-dissolved in 100 μL of anhydrous tetrahydrofuran and azobisisobutyronitrile (AIBN) (initiator, 0.002 mmol) were added sequentially to the above reaction system, and the reaction system was heated to 70 °C and stirred at a constant temperature for 6 h. After the reaction was complete, the crude product was added to 40 mL of anhydrous diethyl ether pre-cooled in an ice bath for precipitation and purification. This process was repeated three times to obtain a yellow viscous product. The residual solvent was removed by vacuum drying, finally yielding the target random copolymer poly(thioctic acid-benzyl acrylate), whose 1H NMR spectrum is shown below. Figure 1 As shown.

[0049] (2) Synthesis of polydisulfide backbone

[0050] The poly(thioctic acid-benzyl acrylate) synthesized in step (1) was azidated to obtain a polydisulfide backbone via an amide bond coupling reaction: 0.4678 mmol of the copolymer was weighed and dissolved in 4 mL of tetrahydrofuran, and 1.5 equivalents of azido-polyethylene glycol 9-amine (0.7 mmol, 338.27 mg) were added; the reaction flask was placed in an ice-water bath, and 4-dimethylaminopyridine DMAP (57.15 mg, 0.4678 mmol, 1 equivalent) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC (179.3 mg, 0.9356 mmol, 2 equivalents) were slowly added; after stirring the reaction system for 10 min, the temperature was slowly raised to room temperature, and the reaction was continued overnight. After the reaction was complete, dichloromethane (DCM) was added to the reaction suspension for dilution, followed by washing with 1M hydrochloric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated saline solution in sequence. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product was added to 40 mL of anhydrous diethyl ether pre-cooled in an ice bath for precipitation and purification. This process was repeated three times. After lyophilization, a light yellow viscous azide-modified random poly(thioctic acid-benzyl acrylate), which is the polydisulfide backbone, was obtained. Its 1H NMR spectrum is shown below. Figure 2 As shown.

[0051] (3) Synthesis of zwitterionic monomer 3-((3-methacrylamidopropyl)dimethylammonium)propionate

[0052] Under nitrogen protection, N-(3-dimethylaminopropyl)methacrylamide (1.7 g, 10 mmol) was dissolved in 150 mL of anhydrous acetone, and the system was kept at 0°C in an ice-water bath. Then, β-propiolactone (715 mg, 10 mmol) was added, and the system was stirred overnight at 5°C. After the reaction was complete, the resulting white precipitate was collected by filtration, washed with diethyl ether, and dried under vacuum to obtain the target monomer 3-((3-methacrylamidopropyl)dimethylammonium)propionate, whose 1H NMR spectrum is shown below. Figure 3 As shown.

[0053] (4) Synthesis of polycarboxylated betaine

[0054] Under a nitrogen atmosphere, 486 mg (2 mmol) of zwitterionic monomer 3-((3-methacrylamidopropyl)dimethylammonium)propionate and 3.725 mg (0.013 mmol) of chain transfer agent 4-cyano-4-(thiobenzoyl)valerate were weighed and dissolved in 1.2 mL of ethanol / water mixture (volume ratio 2:3). After thorough stirring until completely dissolved, 0.00267 mmol of azobisisobutyronitrile (AIBN) was added, and the mixture was stirred at 70 °C overnight. After the reaction was completed, the product was transferred to a dialysis bag and dialyzed against deionized water overnight. After vacuum drying, a pink solid polycarboxylated betaine was obtained, and its 1H NMR spectrum is shown below. Figure 4 As shown.

[0055] (5) Modification of polycarboxylated betaine with dibenzocyclooctylene

[0056] Under a nitrogen atmosphere, the polycarboxylated betaine synthesized in step (4) was dissolved in 1.2 mL of a mixed solvent of ethanol / water (volume ratio 2:3) and stirred until the system was homogeneous. Propylamine (38.42 mg, 53.44 μL, 0.65 mmol) was slowly added, and stirring continued until the reaction solution changed from pink to colorless and transparent. 260 μL (0.13 mmol) of a 0.5 mol / L tris(2-chloroethyl)phosphine aqueous solution was added to the above system. After reacting at room temperature for 1 h, dibenzocyclooctylene-maleimide (27.77 mg), pre-dissolved in 600 μL of dimethyl sulfoxide, was slowly added dropwise to the reaction system, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was dialyzed for 4 h, and the supernatant was collected by centrifugation. After extraction with dichloromethane, the mixture was dialyzed again overnight with deionized water and dried under vacuum to obtain a white solid of dibenzocyclooctylene-modified polycarboxylated betaine. Its 1H NMR spectrum is shown below. Figure 5 As shown.

[0057] (6) Synthesis of brush-structure barrier-adapted nucleic acid drug delivery system All coupling reactions were carried out in aqueous solution via a copper-free azide-alkyne cycloaddition reaction, employing a stepwise grafting strategy to prepare the target delivery system. Taking a system with 70% antisense oligonucleotide content (70% nucleic acid) as an example: antisense oligonucleotides (5 nmol) were added to a water-soluble polydisulfide backbone solution, shaken and mixed, and incubated overnight at 40°C for a copper-free catalytic click chemistry reaction; subsequently, a dibenzocyclooctyne-modified polycarboxybenzene aqueous solution was added to the above reaction system, and incubation continued overnight at 40°C for another copper-free catalytic click chemistry reaction, yielding the target delivery system (70% nucleic acid vector).

[0058] The antisense oligonucleotide sequence is 5′-TTCACGTGTTGCCTGTGGAAAACATGAATT-3′, with DBCO modification at the 5′ T. .

[0059] During the reaction, the molar ratio of the modified antisense oligonucleotide, the polydisulfide backbone, and the dibenzocyclooctylene-modified polycarboxylated betaine was 50:1:22.

[0060] Example 2 The only difference between this embodiment and the brush-structure barrier-adapted nucleic acid drug delivery system in Example 1 is that the target delivery system (30% nucleic acid) with an antisense oligonucleotide content of 30% is prepared by adjusting the feed ratio of each component in the reaction system (the molar ratio of modified antisense oligonucleotide, polydisulfide backbone, and dibenzocyclooctylene-modified polycarboxybetaine is 22:1:50), while keeping other process parameters unchanged, to obtain the target delivery system (30% nucleic acid) carrier.

[0061] Sample Analysis (1) Polymer characterization Figures 1-5 The NMR spectrum results confirm the successful synthesis of the corresponding substance. Among them, Figure 1 and Figure 2 The random polymer (thioctic acid-benzyl acrylate) and polydisulfide backbone were dissolved in DMSO-d6 and characterized by NMR. Figures 3-5 To characterize carboxybetaine monomers, polycarboxybetaine, and dibenzocyclooctylene-modified polycarboxybetaine using D2O.

[0062] (2) Parameter characterization of brush-structure barrier-adapted nucleic acid drug delivery system Figure 6 Potential and hydrated particle size were characterized for 70% and 30% nucleic acid carriers. The potential of the 70% nucleic acid carrier was approximately -7 mV, and that of the 30% nucleic acid carrier was approximately -2 mV. The hydrated particle size of the 70% nucleic acid carrier was approximately 50 nm and that of the 30% nucleic acid carrier (Z-average), respectively. The black marker in the hydrated particle size statistical graph represents the baseline of 0. Both potential and hydrated particle size were obtained using a light scattering instrument nanoparticle size potentiometry at a concentration of 1 mg / mL.

[0063] (3) Enzyme stability test of brush-structure barrier-adapted nucleic acid drug delivery system Figure 7 The results characterize the nuclease stability of a 30% nucleic acid vector. The experiment used fetal bovine serum containing 20% ​​fetal bovine serum to simulate the degradation of nucleic acids by various nucleases, with a nucleic acid concentration of 100 nM. The results show that the brush-structure barrier-adapted nucleic acid drug delivery system can significantly improve the enzymatic stability of nucleic acid drugs, extending the degradation half-life by more than 50 times.

[0064] (4) Cell uptake experiment of brush-structure barrier-adapted nucleic acid drug delivery system Figure 8 Figures AB in the diagram show the uptake of 70% and 30% of the nucleic acid carrier by mouse bone marrow-derived mast cells. The nucleic acid concentration was 100 nM, and the incubation time was four hours. Unuptaken nucleic acids were then washed away with PBS, and cellular uptake was characterized. The results indicate that the brush-like barrier-adapted nucleic acid drug delivery system can significantly improve the uptake of nucleic acid drugs by cells.

[0065] (5) Cytotoxicity assays of brush-structure barrier-adapted nucleic acid drug delivery systems Figure 9 The graph shows the cytotoxicity analysis of 70% and 30% nucleic acid vectors against human umbilical vein endothelial cells, mouse fibroblasts, and mouse bone marrow-derived mast cells. The nucleic acid concentrations ranged from 0.1 to 10 μM, and the incubation time with cells was 72 hours. This indicates that the brush-like barrier-adapted nucleic acid drug delivery system is essentially non-toxic.

[0066] (6) Therapeutic effect of brush-structure barrier-adapted nucleic acid drug delivery system on mice with atopic dermatitis A brush-like barrier-adapted nucleic acid drug delivery system (70% nucleic acid carrier) prepared using the above method was used for transdermal treatment of atopic dermatitis with antisense oligonucleotides (5′-TTCACGTGTTGCCTGTGGAAAACATGAATT-3′, 5′ modified with DBCO). After constructing a mouse-specific dermatitis model using toluene diisocyanate, the (70% nucleic acid carrier) was applied to the left ear of mice (drug administration group), with a nucleic acid quantification of 1 nmol. The right ear (control group) was treated with a matrix ointment for 7 days. On the final challenge day, the thickness of both ears was measured using a micrometer as a baseline. Then, 20 μL of 1% toluene diisocyanate solution was applied to both ears to induce large-scale degranulation of mast cells in sensitized mice. Twenty-four hours after challenge, the ear thickness was measured again, and the swelling change was calculated. Mice were then sacrificed, and ear tissue was harvested for subsequent analysis. The core characterization indicators and methods for atopic dermatitis are as follows: Edema index was measured using a digital micrometer, in μm, reflecting the severity of inflammation. Mice were lightly anesthetized during measurement, avoiding the cartilaginous prominence at the base of the ear. Each ear was measured independently three times, and the average value was taken. FcεRI receptor expression was verified using immunohistochemical sections to verify the experimental mechanism of action.

[0067] Figure 10 This study investigated the skin penetration depth of a 70% nucleic acid carrier for nucleic acid drug treatment of atopic dermatitis. Results showed that the 70% nucleic acid carrier significantly penetrated the skin, reaching a depth of 200 μm. In contrast, free nucleic acids could barely penetrate the skin barrier. These results fully demonstrate that the brush-structured barrier-adapted nucleic acid drug delivery system prepared in this invention can overcome the skin barrier.

[0068] Figure 11 This study investigated the efficacy of a 70% nucleic acid vector for nucleic acid drug treatment of atopic dermatitis. Results showed that the brush-like barrier-adapted nucleic acid drug delivery system effectively penetrated the skin barrier, demonstrating significant intervention effects in treating atopic dermatitis. There was no significant difference in ear thickening between the treated and control groups. Immunohistochemical results indicated a significant downregulation of the target protein after treatment with the 70% nucleic acid vector.

[0069] (7) Therapeutic effect of brush-structure barrier-adapted nucleic acid drug delivery system on mice with allergic rhinitis A brush-like barrier-adapted nucleic acid drug delivery system (30% nucleic acid carrier) prepared using the above method was used to prevent and treat allergic rhinitis with antisense oligonucleotides. Allergic rhinitis was induced in mice using aluminum hydroxide and chicken ovalbumin. The mice were then administered the drug via nasal drops at a nucleic acid quantification of 1 nmol for 7 days. On the final challenge day, chicken ovalbumin was used to induce allergic rhinitis, and allergic physiological phenomena, including rhinorrhea and sneezing, were observed. Blood was collected from viable mice via facial / submandibular veins at 15 minutes, and serum was separated for histamine content detection. The mice were sacrificed the following day, and head tissue was dissected, with the nasal tip used for histopathological examination. Serum histamine levels were measured, and PAS staining was used to detect goblet cell metaplasia in the nasal septum respiratory epithelium.

[0070] Figure 12 This study investigated the mucus penetration of a 30% nucleic acid carrier for nucleic acid drug treatment of allergic rhinitis. Results showed that the diffusion rate of the 30% nucleic acid carrier in mucus (2% porcine gastric mucin PBS solution) was significantly higher than that of free nucleic acid, with a mean square displacement approximately 120 times that of free nucleic acid within 20 seconds. This result fully demonstrates that the brush-structure barrier-adapted nucleic acid drug delivery system prepared in this invention can overcome the mucus barrier in the respiratory tract.

[0071] Figure 13 This study investigated the nasal mucosal penetration of a 30% nucleic acid carrier for nucleic acid drug treatment of allergic rhinitis. Results showed that the 30% nucleic acid carrier significantly penetrated the nasal mucosa, reaching a penetration depth of 150 μm. In contrast, free nucleic acids could hardly penetrate the nasal mucosal barrier. These results fully demonstrate that the brush-structured barrier-adapted nucleic acid drug delivery system prepared in this invention can overcome the nasal mucosal barrier.

[0072] Figure 14The study in section AF investigated the efficacy of a 30% nucleic acid vector for the treatment of allergic rhinitis with nucleic acid drugs. Results showed that the 30% nucleic acid vector effectively crossed the respiratory barrier, demonstrating a significant intervention effect on allergic rhinitis. Allergy-related physiological activities showed no significant difference between the treated group and the control group. Immunohistochemical results showed significant downregulation of the target protein. The number of nose wipings and sneezes in mice was counted individually for each mouse within 15 minutes after stimulation. Nasal discharge was determined by observing the mice's noses with paper. Periodic acid-Schiff staining results indicated severe damage to mucosal goblet cells in the model group, while the 30% nucleic acid vector treatment group showed almost no difference from the control group.

[0073] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A brush-like barrier-adapted nucleic acid drug delivery system, characterized in that, It is prepared by a click reaction or cycloaddition reaction of a polydisulfide backbone, a modified nucleic acid drug, and a modified zwitterion; The polydisulfide backbone is a random copolymer, including disulfide structural unit A and acrylate structural unit B. Disulfide structural unit A is formed by ring-opening polymerization of thioctic acid and modified with click reaction functional group or cycloaddition reaction functional group R3. The modified nucleic acid drug contains 1-100 bases, at least one base is modified with a click reaction functional group or a cycloaddition reaction functional group R4, which can react with R3 of the disulfide structural unit A in a click reaction or cycloaddition reaction. The modified zwitterionic structure is as follows: ； R5 is a click reaction functional group or a cycloaddition reaction functional group, which can react with R4 modified on the nucleic acid drug in a click reaction or a cycloaddition reaction; R6 is a zwitterionic functional group, where m or n is an integer from 1 to 500; and R7 is a chain transfer reagent residue.

2. The brush-structure barrier-adapted nucleic acid drug delivery system according to claim 1, characterized in that, In the polydisulfide backbone, acrylate structural unit B is a benzyl acrylate unit, a 2,2,2-trifluoroethyl acrylate unit, a linear fatty alcohol acrylate unit, an isobornyl acrylate unit, or a polyethylene glycol acrylate unit.

3. The brush-structure barrier-adapted nucleic acid drug delivery system according to claim 1, characterized in that, The structural formula of the polydisulfide backbone is as follows: ； Where x is an integer from 1 to 500, y is an integer from 1 to 2000, and z is an integer from 1 to 1000; R1 and R1' are chain transfer reagent residues, R2 is an acrylate side group, and R3 is selected from any of the following structural formulas. Indicates the connection position; 。 4. The brush-structure barrier-adapted nucleic acid drug delivery system according to claim 1, characterized in that, The R4 modified on the nucleic acid drug is selected from any of the following structural formulas. Indicates the connection position; 。 5. The brush-structure barrier-adapted nucleic acid drug delivery system according to claim 1, characterized in that, In the modified zwitterion, R5 is selected from any of the following structural formulas. Indicates the connection position; 。 6. The brush-structure barrier-adapted nucleic acid drug delivery system according to claim 1, characterized in that, In the modified zwitterion, R6 is selected from any of the following structural formulas. Indicates the connection position; 。 7. The brush-structure barrier-adapted nucleic acid drug delivery system according to claim 1, characterized in that, When polydisulfide backbone, modified nucleic acid drug and modified polyzwitterion react, the molar ratio is 1:0.01-1000:0.01-10000.

8. The method for preparing a brush-like barrier-adaptor nucleic acid drug delivery system according to any one of claims 1-7, characterized in that, Includes the following steps: In a solvent system, a click reaction or cycloaddition reaction is carried out between a polydisulfide backbone and one or more modified nucleic acid drugs, followed by the addition of one or more modified zwitterions to carry out a click reaction or cycloaddition reaction, thereby preparing the brush-like barrier-adapted nucleic acid drug delivery system. When a click reaction occurs, the reaction conditions are 0-60 °C for 2-48 h; When a cycloaddition reaction occurs, the reaction conditions are 25-100 °C for 1-72 h.

9. The method for preparing the brush-like barrier-adapted nucleic acid drug delivery system according to claim 8, characterized in that, The synthesis method of polydisulfide backbone is as follows: under the action of an initiator, thioctic acid, acrylate monomers and chain transfer reagents are polymerized in a solvent system to prepare the first intermediate; under the action of a catalyst, the first intermediate is reacted with a compound containing R3, and after the reaction is completed, the polydisulfide backbone is prepared by post-treatment. And / or, the modified polyzwitterion is prepared by reacting a chain transfer reagent with a zwitterionic monomer under the action of an initiator to obtain a second intermediate; after the second intermediate is dethiocarbonylated by n-propylamine, it undergoes an addition reaction with a methacrylate or maleimide compound containing an R5 group to prepare the modified polyzwitterion.

10. The use of the brush-structure barrier-adaptor nucleic acid drug delivery system according to any one of claims 1-7 in the preparation of disease treatment products and / or disease prevention products.

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